Disturbed Prefrontal Cortex Activity in the Absence of Schizophrenia-Like Behavioral Dysfunction in Arc/Arg3.1 Deficient Mice

The Journal of Neuroscience, October 9, 2019 • 39(41):8149–8163 • 8149

Behavioral/Cognitive Disturbed Prefrontal Cortex Activity in the Absence of Schizophrenia-Like Behavioral Dysfunction in Arc/Arg3.1 Deficient Mice

X Xiaoyan Gao,1 Jasper Grendel,1 Mary Muhia,2 Sergio Castro-Gomez,1 Ute Su¨sens,1 XDirk Isbrandt,3,4,5 Matthias Kneussel,2 XDietmar Kuhl,1 and XOra Ohana1 1Institute for Molecular and Cellular Cognition, 2Institute for Molecular Neurogenetics, 3Experimental Neuropediatrics, Center for Molecular Neurobiology Hamburg, University Medical Center Hamburg–Eppendorf, 20251 Hamburg, Germany, 4Institute for Molecular and Behavioral Neuroscience, University of Cologne, 50937 Cologne, Germany, and 5Experimental Neurophysiology, German Center for Neurodegenerative Diseases, 53175 Bonn, Germany

Arc/Arg3.1, an activity regulated immediate early gene, is essential for learning and memory, synaptic plasticity, and maturation of neural networks. It has also been implicated in several neurodevelopmental disorders, including schizophrenia. Here, we used male and female constitutive and conditional Arc/Arg3.1 knock-out (KO) mice to investigate the causal relationship between Arc/Arg3.1 deletion and schizophrenia-linked neurophysiological and behavioral phenotypes. Using in vivo local field potential recordings, we observed damp- ened oscillatory activity in the prefrontal cortex (PFC) of the KO and early conditional KO (early-cKO) mice, in which Arc/Arg3.1 was deleted perinatally. Whole-cell patch-clamp recordings from neurons in PFC slices revealed altered synaptic properties and reduced network gain in the KO mice as possible mechanisms underlying the oscillation deficits. In contrast, we measured normal oscillatory activity in the PFC of late conditional KO (late-cKO) mice, in which Arc/Arg3.1 was deleted during late postnatal development. Our data show that constitutive Arc/Arg3.1 KO mice exhibit no deficit in social engagement, working memory, sensorimotor gating, native locomotor activity, and dopaminergic innervation. Moreover, adolescent social isolation, an environmental stressor, failed to induce deficits in sociability or sensorimotor gating in adult KO mice. Thus, genetic removal of Arc/Arg3.1 per se does not cause schizophrenia-like behavior. Prenatal or perinatal deletion of Arc/Arg3.1 alters cortical network activity, however, without overtly disrupting the balance of excitation and inhibition in the brain and not promoting schizophrenia. Misregulation of Arc/Arg3.1 rather than deletion could potentially tip this balance and thereby promote emergence of schizophrenia and other neuropsychiatric disorders. Key words: Arc/Arg3.1; excitation/inhibition; knock-out; local field potential; prefrontal cortex; schizophrenia

Significance Statement The activity-regulated and memory-linked gene Arc/Arg3.1 has been implicated in the pathogenesis of schizophrenia, but direct evidence and a mechanistic link are still missing. The current study asks whether loss of Arc/Arg3.1 can affect brain circuitry and cause schizophrenia-like symptoms in mice. The findings demonstrate that genetic deletion of Arc/Arg3.1 before puberty alters synaptic function and prefrontal cortex activity. Although brain networks are disturbed, genetic deletion of Arc/Arg3.1 does not cause schizophrenia-like behavior, even when combined with an environmental insult. It remains to be seen whether misregulation of Arc/Arg3.1 might critically imbalance brain networks and lead to emergence of schizophrenia.

Introduction ing delusions and hallucinations, negative symptoms, including Schizophrenia is a debilitating neuropsychiatric disease with a social withdrawal and anhedonia, and cognitive symptoms re- high penetration (1%) of the population and a strong hereditary flecting deficits in working memory and executive control (van component. Although heterogeneous, the core behaviors charac- Os and Kapur, 2009; Owen et al., 2016). The neurophysiological terizing schizophrenia are divided in positive symptoms, includ- This work was supported by the Grant “Molekulare Mechanismen der Netzwerkmodifizierung” to D.K. and O.O. from the Federal State of Hamburg, by a Grant from the Schaller-Nikolich Foundation to D.K. and O.O., by the DFG Received March 19, 2019; revised Aug. 6, 2019; accepted Aug. 29, 2019. GrantKN556/11-2(FOR2419)toM.K.X.G.wassupportedbyascholarshipfromtheChinesegovernmentawardedby Author contributions: X.G., D.K., and O.O. designed research; X.G., J.G., M.M., S.C.-G., U.S., D.I., and O.O. per- theChinaScholarshipCouncil.WethankSabineHoffmeister-UlrichforperformingCNVonCrerecombinasecarrying formed research; D.K. contributed unpublished reagents/analytic tools; X.G., J.G., M.M., S.C.-G., D.I., M.K., D.K., and mice and to Peter Soba for assistance with Imaris. Confocal images were taken in the Microscopy Imaging Facility of O.O. analyzed data; X.G., D.K., and O.O. wrote the paper. the University Medical Center Hamburg–Eppendorf with technical support from Antonio Virgilio Failla. 8150 • J. Neurosci., October 9, 2019 • 39(41):8149–8163 Gao et al. • Arc/Arg3.1 Involvement in Schizophrenia characteristics of the disease include altered brain activity pat- Arc/Arg3.1 KO mice in three benchmark tests of schizophrenia in terns and uncoordinated inter-areal communication (Lisman, rodents. Moreover, given the longstanding theory about the dys- 2012; Uhlhaas and Singer, 2015; Hunt et al., 2017). Although function of the dopamine system in schizophrenia (Toda and psychosis commonly manifests during puberty and early adult- Abi-Dargham, 2007; Howes and Kapur, 2009; Howes et al., hood, cognitive and neurophysiological phenotypes can be 2015), we examined the sensitivity to dopamine elevation and the detected earlier, which has led to the prevalent view of schizo- dopaminergic innervation of the PFC and striatum. In humans, phrenia as a neurodevelopmental disorder (Rapoport et al., 2012; schizophrenia has a high rate of comorbidity with epilepsy Birnbaum and Weinberger, 2017). This view holds that the pro- (Ma¨kikyro¨ et al., 1998; Qin et al., 2005; Clarke et al., 2012), which cess of normal brain development is interrupted by polygen- is recapitulated in several animal models of the disease (Fejgin et etic and environmental factors (van Os and Kapur, 2009; al., 2014). To investigate a possible association between Arc/ Modai and Shomron, 2016; Owen et al., 2016), which ulti- Arg3.1 deletion, epileptic activity, and schizophrenia, we per- mately alter neurotransmitter systems, brain connectivity, and formed electrocorticogram recordings from chronically implanted cognitive functions. WT and KO mice while monitoring their activity over several The activity-regulated gene Arg3.1 (Link et al., 1995), also days. Finally, we tested the interaction between Arc/Arg3.1 dele- known as Arc (Lyford et al., 1995), plays an essential role in adult tion and an environmental stressor on schizophrenia-like learning and memory and synaptic plasticity (Guzowski et al., behaviors. 2000; Plath et al., 2006; Messaoudi et al., 2007; Jakkamsetti et al., 2013; El-Boustani et al., 2018; Gao et al., 2018). Additionally, Arc/Arg3.1 also mediates forms of plasticity that are prominent Materials and Methods during development, such as homeostatic synaptic scaling Mice. Mice aged 3–5 months were kept in a vivarium with an inverted (Chowdhury et al., 2006; Shepherd et al., 2006), metabotropic 12 h light/dark cycle (08:00–20:00 dark period) in groups, under stan- Ϯ glutamate receptor-dependent long-term depression (Waung et dard housing conditions (23 1°C, 40–50% humidity; food and water al., 2008) and synapse elimination (Mikuni et al., 2013). Arc/ ad libitum). Animals were housed together in groups of 3–5 mice per cage. Mice subjected to social isolation were housed individually from Arg3.1 upregulation during early postnatal development shapes postnatal day (P)35 until the day of the experiment. Both male and hippocampal oscillatory activity and learning capacity (Gao et al., female mice were used in all experiments. All procedures were conducted 2018), likely by changing network connectivity and microarchi- in accordance with the German and European Community laws on pro- tecture. This may also explain why misregulation of Arc/Arg3.1 is tection of experimental animals and approved by the local authorities of linked to neurodevelopmental diseases, such as fragile X Syn- the City of Hamburg. Experimenters were blind to the genotype until the drome (Niere et al., 2012; Ronesi et al., 2012) and Angelman conclusion of experiments and analysis. syndrome (Greer et al., 2010; Cao et al., 2013; Mandel-Brehm et Generation of constitutive and conditional Arc/Arg3.1 KO mice. Arc/ al., 2015). Previous studies on schizophrenia patients implicated Arg3.1 KO mice were generated as previously described (Plath et al., Arc/Arg3.1 as a molecular hub in synaptic gene networks whose 2006). Along with the KO mice generation, floxed Arc/Arg3.1 mutants f/f abnormalities possibly play a significant role in the pathogenesis (Arc/Arg3.1 ) were generated in parallel and thoroughly validated for of schizophrenia (Kirov et al., 2012; Fromer et al., 2014; Purcell et use in biochemical and behavioral experiments (Gao et al., 2018). Con- ditional Arc/Arg3.1 gene ablation in the brain was accomplished by al., 2014; Ferna´ndez et al., 2017). In addition, a recent study breeding Arc/Arg3.1f/f with the Cre recombinase transgenic mice (1) (Manago` et al., 2016) reported schizophrenia-like behaviors and Tg(CaMKII␣-cre)1Gsc (Casanova et al., 2001) to obtain early condi- dysregulated dopaminergic transmission in Arc/Arg3.1-deficient tional KO (early-cKO) mice, and (2) Tg(CaMKII␣-cre)T29–1Stl (Tsien mice (Wang et al., 2006). However, the presence of a neomycin et al., 1996) to obtain late conditional KO (late-cKO) mice. Breeding was cassette and an EGFP sequence in the genome of these mice can performed in accordance with recommended procedure for LoxP-Cre result in artifactual aberrations in gene expression and confound recombination (Song and Palmiter, 2018). Briefly, in generation F1 Arc/ the conclusions of the study. Arg3.1f/f mice were crossed with Arc/Arg3.1ϩ/ϩ,Creϩ mice. In generation ϩ ϩ ϩ Here, we used independently generated constitutive and con- F2 Arc/Arg3.1f/ ,Cre were crossed with Arc/Arg3.1f/ to obtain cKO ϩ ϩ ϩ ϩ ditional Arc/Arg3.1 knock-out (KO) mice to ask whether loss of mice. Mice with genotypes Arc/Arg3.1 / ,Cre and Arc/Arg3.1f/f,Cre Arc/Arg3.1 by itself is indeed sufficient to evoke neurophysiolog- were used for experiments as WT controls and cKOs, respectively. Mice ical and behavioral dysfunctions of schizophrenia. Alterations in were genotyped by PCR for Arc/Arg3.1 and by PCR and copy number cortical activity, in particular, aberrant gamma oscillations (Lis- variation for Cre. Genotyping was performed between P5 and P7 and again after termination of the experiment. man, 2012; Uhlhaas and Singer, 2015; Hunt et al., 2017) are con- In vivo LFP recording in the PFC. LFP recording and analysis were sidered an endophenotype of schizophrenia and can be observed performed as previously described (Prox et al., 2013; Fazeli et al., 2017). in prodromal stages of the disease, before manifestations of be- In short, adult male mice were injected with 0.8 mg/g body weight ure- havioral symptoms. Hence, we recorded local field potential thane (10% w/v, in 0.9% sodium chloride; Sigma-Aldrich) and 0.05 ␮g/g (LFP) in the prefrontal cortex (PFC) of anesthetized WT and KO body weight of buprenorphine (Temgesic). Initial anesthesia was in- mice and compared these to conditional KO mice in which Arc/ duced with 4% isoflurane, which was decreased to 1.0–1.5% isoflurane Arg3.1 was deleted after late postnatal development. We labeled during surgery. A linear 16-site silicon probe with a 100 ␮m inter- synaptic proteins in the PFC and measured glutamatergic and electrode distance and 177 ␮m 2 electrode surface (a1x16-5 mm-100– GABAergic neurotransmission in PFC neurons to elucidate syn- 177; NeuroNexus Technologies) was vertically lowered into the PFC of aptic mechanisms underlying aberrant network activity and cog- the right hemisphere (1.5 mm anterior to bregma, 0.3 mm lateral to nitive dysfunction. In addition, we assessed the behavior of adult midline, 3.3 mm deep). Data from the probe was digitally filtered (0.5– 9000 Hz bandpass) and digitized as 16-bit integers with a sampling rate of 32 kHz using a Digital Lynx 4SX data acquisition system (NeuraLynx). During the recording, anesthetic depth was monitored from breathing The authors declare no competing financial interests. Correspondence should be addressed to Ora Ohana at [email protected] or Dietmar Kuhl at rates, twitching and electrophysiological properties. If required, addi- [email protected]. tional 0.2 mg/g body weight urethane doses were given. The position of https://doi.org/10.1523/JNEUROSCI.0623-19.2019 the probe, dipped in a DiI-solution before insertion, was verified post- Copyright © 2019 the authors mortem from NeuroTrace fluorescent DAPI-stained (Invitrogen) coro- Gao et al. • Arc/Arg3.1 Involvement in Schizophrenia J. Neurosci., October 9, 2019 • 39(41):8149–8163 • 8151

nal slices. Analysis was performed from electrodes located in layer (L)5 of were visually inspected by the experimenter. Traces of PSCs evoked by the infralimbic (IL) region of the PFC. repeated stimuli (3–6 per stimulus intensity) were baseline subtracted All in vivo data were analyzed and visualized in MATLAB (Math- and averaged. At each stimulus intensity, the averaged eEPSC trace Works) or NeuroScope (Hazan et al., 2006). LFPs were downsampled to evoked under gabazine was subtracted from the ePSC trace recorded 1.28 kHz from raw traces. Multitaper time-frequency spectra were com- under control ACSF to obtain a hypothetical evoked IPSC trace (eIPSC). puted using Chronux (http://chronux.org). One power spectrum per The charge was calculated from the averaged ePSC, eEPSC, and eIPSC mouse was calculated from concatenated epochs (nFFT ϭ 4096, nw ϭ 1). traces by integrating the current over an identical time window begin- Theta (3.8–6 Hz) and gamma (20–50 Hz) power was calculated for ning 3 ms after stimulus and ending when the longer trace returned to selected rapid eye movement (REM)-like epochs by integrating the area baseline. The excitation/inhibition (E/I) ratio of the network was calcu- below the theta and gamma ranges. lated by dividing the eEPSC charge carried by eIPSC. Experiments were In vitro recordings in PFC slices. Mice, 3–4 months old, were anesthe- included in the analysis, which contained a complete series of ePSC re- tized with isoflurane and decapitated. Brains were rapidly removed and cordings at all current steps between 15 and 50 ␮A (see Fig. 3D,E) in both immersed in ice-cold slicing artificial CSF (slicing-ACSF), continuously ACSF and gabazine. ␮ gassed with a mixture of 95% O2,5%CO2. Coronal slices (350 m) were Adolescent social isolation. WT and Arc/Arg3.1 KO mice were socially cut (Microme 650H vibratome, ThermoFisher Scientific), immediately isolated by individual housing from P35 and onward. Behavioral tests transferred into a holding chamber containing oxygenated warmed were conducted when the mice were 3 months old (Niwa et al., 2013; Li et recording-ACSF and allowed to recover for 1–4 h at 36°C. Slicing-ACSF al., 2018).

contained the following (in mM): 212 sucrose, 2.6 KCl, 1 CaCl2, 3 MgSO4, Sociability and social novelty tests. This test was done in a three- ϫ 1.25 NaH2PO4, 26 NaHCO3, 10 glucose. Recording-ACSF contained the chambered arena made of transparent Plexiglas (20 38.5 cm for the left ϫ following (in mM): 119 NaCl, 2.5 KCl, 2.5 CaCl2, 1.3 MgSO4, 1.25 and right side chambers, 12 38.5 cm for the middle chamber). Plexiglas NaH2PO4, 26 NaHCO3, 10 glucose, and was maintained at 37°C and walls separated the three chambers, which were connected by open doors pH 7.4. in the walls. Two transparent plastic cylinders with holes were placed in Recordings were made in a submerged chamber continuously per- the left and right chamber. During the habituation phase, each mouse fused with oxygenated recording-ACSF (3 ml/min) maintained at 35– was released in the middle chamber and allowed to explore the arena for 37°C. Slices containing the PFC (bregma coordinates 1.4–2.2) were 10 min every day for 2 d. In the sociability test, a Dummy Lego mouse and visualized with an inverted Olympus BX51WI microscope. A 4ϫ objec- Novel Mouse 1 were placed underneath a plastic cylinder separately. A tive was used to localize L5 in the IL or prelimbic (PrL) regions of the PFC weighted object was placed on top to prevent the mice from climbing to and to place a stimulating electrode (concentric stainless steel, 0.1 M⍀ the top or toppling the cylinder. The test mice were placed into the impedance) in L1 of the same region. Neurons in L5 were visualized and middle chamber for 10 min of free exploration. Two zones, centered on targeted for patching with infrared differential interference contrast mi- either cylinder with a radius 1.5 cm larger than the cylinders were defined croscopy at 63ϫ magnification. Pipettes (5–8 M⍀) were pulled from as the close interaction zones. The duration the test mouse spent within borosilicate glass and filled with a recording solution containing the these zones and in each chamber was recorded. The location of the following (in mM): 115 potassium gluconate, 4 KCl, 10 HEPES, 10 Dummy Lego mouse or Novel Mouse1 was pseudorandomized. Subse-

phosphocreatine-Na4 ATP-Mg, 0.3 guanosine triphosphate, and 0.5% quently, the same test mouse was subjected to the social novelty test. In biocytin. Osmolarity was adjusted to 280–290 mOsm with KOH. This this test, the Dummy Lego mouse was replaced by Novel Mouse 2, and solution was chosen to increase the driving force for Cl Ϫ currents (cal- Novel Mouse 1 was kept in the same cylinder as in the sociability test. The culated as Ϫ82 mV) and facilitate detection of spontaneous IPSCs test mouse was again released into the middle chamber and allowed to (sIPSCs) Somatic whole-cell patch-clamp recordings were made in explore for another 10 min. The duration the test mouse spent within the current- and voltage-clamp modes with a MultiClamp 700b amplifier close interaction zones and duration spent in exploring the three cham- (Molecular Devices). Signals were sampled at 10 kHz and filtered (3 kHz) bers was recorded. All the novel mice used in the experiments are wild using Digidata 1422 (Molecular Devices). Series resistance (range 12–40 types with gender and genetic background the same as the test mice. M⍀) was monitored continuously but compensated only in the current Videos were recorded, and mice exploration behaviors were tracked with clap mode. Experiments where Rs changed Ͼ30% were excluded from EthoVision XT (Noldus Information Technology). Ethanol (35%) was analysis Data were collected and visualized using the pCLAMP 10 soft- used for cleaning the arena between subjects. ware (Molecular Devices), exported and analyzed off-line, using Clamp- Y-maze continuous spontaneous alternation. The Y-maze apparatus fit10 and IGOR Pro 6.3 (WaveMetrics). A typical experiment consisted of consisted of three identical arms (length ϫ width ϫ height: 37.5 ϫ 7.5 ϫ a series of hyperpolarizing and depolarizing currents steps in current- 16 cm) made of opaque beige Plexiglas and interconnected at 120° from clamp, to determine the input resistance (Rin) of the cell and its action a central triangle. It was located in a room with diffuse lighting (40 lux in potential firing pattern and frequency. Subsequently, neurons were volt- each arm). For each trial, mice were placed in one arm of the maze and age clamped at membrane potential of Ϫ70 and Ϫ50 mV to measure allowed to explore freely for 5 min during which the number and se- spontaneous EPSCs (sEPSCs) and sIPSCs, respectively. sIPSCs recorded quence of arm entries were noted down. A total number of arm entries at Ϫ50 mV were consistently abolished following application of the was used as a measure for activity. The number of alternations, defined as ␮ GABAA antagonist gabazine (10 M; Abcam). To measure evoked PSCs successive entries into each of the three arms as an overlapping triplet set (ePSCs), neurons were clamped to Ϫ70 mV and a series of incremental was calculated. Percentage alternation scores were then computed as current steps (0.1 ms, 10–50 ␮A) was applied by a stimulus isolator follows: spontaneous alternation (%) ϭ [(Number of alternations)/(To- (Iso-Flex, AMPI) to the extracellular electrode in L1. Finally, gabazine tal number of arm entries Ϫ 2)] ϫ 100. (10 ␮M) was added to the ACSF and recordings of the sEPSC, sIPSCs, and Prepulse inhibition of the acoustic startle reflex. Prepulse inhibition ePSCs were repeated. At the end of experiments, slices were fixed in 4% (PPI) of the acoustic startle reflex was examined in two startle response paraformaldehyde (PFA) and processed, as previously published (Ohana systems (SR-LAB, San Diego Instruments). The test platform consisted et al., 2012), to reveal the biocytin in the recorded neurons. Stained of a transparent cylindrical mouse enclosure mounted on a Plexiglas base neurons were carefully examined at the light microscope and their den- and an accelerometer sensor connected at the base. This was housed dritic morphology and laminar and regional locations were noted. Only inside a sound-attenuating chamber containing a light source and a neurons with typical pyramidal morphology in L5 were selected for sound generation system that emitted continuous background white analysis (van Aerde and Feldmeyer, 2015). Few neurons were 3-di- noise (65 dB) and the various acoustic stimuli used during the test ses- mensionally reconstructed at a 40ϫ magnification with the Neurolucida sion. Before testing, startle calibration, and chamber standardization was system (MBF Bioscience). conducted by mounting an SR-LAB Standardization Unit (San Diego Data analysis: for sEPSC and sIPSC analysis, current traces were fil- Instruments) onto the animal enclosures to attain a reasonable waveform tered off-line at 1 kHz and sEPSCs and sIPSCs were detected automati- response (700 Ϯ 15 mV) and to ensure that a given baseline response was cally in Clampfit at a threshold of Ϫ5 and 5 pA, respectively. All events similar for both test chambers. 8152 • J. Neurosci., October 9, 2019 • 39(41):8149–8163 Gao et al. • Arc/Arg3.1 Involvement in Schizophrenia

The test session began by introducing the mice in the animal enclo- intensity image. The mean TH immunofluorescence was measured from sures. Following a 5 min acclimation period, mice were first presented regions-of-interest (ROIs) positioned over the striatum or PFC and nor- with six pulse-alone trials to habituate and stabilize their startle response. malized to the ROI area. The animals were subsequently presented with 12 main blocks of trials, Synaptic clusters were quantified in the PrL and IL regions of the PFC each comprising a mixture of 12 discrete trial types: pulse-alone trials, from three WT and three KO mice. Three brain sections per mouse were prepulse-plus-pulse trials, prepulse-alone trials, and trials in which no taken at bregma coordinates 1.7–1.9. Non-overlapping image stacks (12 discrete stimulus other than constant background noise was presented. per mouse) were captured in the cell-sparse and dendrites-enriched L1, The discrete trial types within each block were presented in pseudoran- using a 63ϫ 1.4 NA oil-immersion objective and a 2.4ϫ digital zoom dom order with variable intertrial intervals ranging from 10 to 20 s. All with the pinhole set to 1 AU. Stacks of four consequent images were stimuli were in the form of white noise with a rise time of ϳ1 ms pre- captured at 1024 ϫ 1024 pixels and 0.25 ␮m z-steps increment, resulting sented against a constant background noise level of 65 dB. Pulse stimuli in imaged region size of 76.88 ϫ 76.88 ϫ 1 ␮m and a voxel size of 0.075 ϫ were 40 ms in duration and 120 dB in intensity. Prepulse stimuli were 20 0.075 ϫ 0.25 ␮m. Laser intensity, detector sensitivity and line averaging ms in duration and were 69, 73, 77, 81, or 85 dB in intensity. This corre- were optimized against sections stained with secondary antibodies only. sponded to ϩ4, ϩ8, ϩ12, ϩ16, or ϩ20 dB units above background noise. Identical image acquisition parameters were applied to the sections of all There were, therefore, five possible combinations of prepulse-plus-pulse mice. Image stacks were analyzed in Imaris 9.3 (Bitplane) using the Spot trials, for which a stimulus onset asynchrony of 100 ms between the two function and the MATLAB R2017 (MathWorks) extension for Spot co- stimuli was used. The test session concluded with the presentation of six localization. Images were background subtracted. Spots were automati- pulse-alone trials and lasted ϳ48 min. cally detected above a threshold of 0.2 ␮m diameter for PSD-95 and Whole-body responses were transduced by the accelerometer sensor, gephyrin and 0.3 ␮m for synaptophysin. Colocalization between synap- digitized, and transmitted to a computer running the SR-LAB software. tophysin and either gephyrin or PSD-95 spots was set at a maximal Startle reaction was analyzed by measuring the maximum (Vmax) of the center-to-center distance of 0.7 ␮m. waveform response (from 0 to 65 ms) to the stimulus for every trial. Kainic acid-induced seizures. Mice were taken from their cages and Percentage of PPI for each prepulse trial (69, 73, 77, 81, or 85 dB) was received an intraperitoneal injection of 20 ␮g kainic acid per gram body calculated according to the following formula: percentage PPI ϭ [(mean weight. From there on animals were monitored for seizure symptoms. startle response to pulse alone trials) Ϫ (mean startle response for Seizures were defined as having occurred when the animals showed typ- prepulse-plus-pulse trials)/(mean startle response to pulse alone trials)] ϫ ical symptoms of rearing with forelimb clonus, falling, and rotating. If 100. animals did not show any of these symptoms within 30 min, they were Locomotor activity and amphetamine sensitivity test in the open field. administered additional equal or lower dosages of kainic acid until sei- Spontaneous exploration and locomotor activity were evaluated in an zures occurred. The seizure strength was scored by the following criteria: open-field arena (50 ϫ 50 ϫ 50 cm) constructed from opaque white (1) Strong seizure activity like clonus of forelimbs and head, rearing but forex plates. In this test, mice are allowed to freely explore the arena for no falling; (2) as in 1, but additionally falling; (3) as in 2, but more severe 1 h. Locomotor activity was video-recorded under dim indirect illumi- with additional jumping, uncontrolled circling or repetitive falling. The nation (40 lux) by using EthoVision XT video-tracking program (Noldus final dose of kainic acid injected (␮g/g body weight) was calculated from Information Technology). To test the dopamine sensitivity, an indepen- the sum of all injection volumes. dent group of mice was injected with D-amphetamine intraperitoneally Electrocorticogram recording and analysis. Data were collected as pre- (1.5–2.5 mg/kg body weight, Fagron). Locomotor activity was recorded viously described (Marguet et al., 2015). Telemetric electrocorticogram for 10 min before injection and for 1 h after the injection. For all tests, the analyses were performed using implantable radio transmitters (models path length and velocity were analyzed. The arena was cleaned with 70% TA11EA-F20 or TA11ETA-F20, DataSciences International). Adult (at ethanol between subjects. least 3 months old) male mice received 0.05 mg/kg buprenorphine (sub- Immunofluorescence staining and confocal imaging. Adult mice were cutaneously) for analgesia and were anesthetized with 1.5–2% isoflurane deeply anesthetized and transcardially perfused with 0.1 M PBS followed in 100% oxygen. Midline skin incisions were made on the top of the skull by 4% PFA. Brains were removed, postfixed with 4% PFA for 2–6 d, and in the neck. The transmitter body was implanted subcutaneously. cryoprotected in 30% sucrose for 2 d, embedded in Tissue-Tek OCT The tips of the leads were placed 2 mm posterior to bregma and 1.8 mm (Sakura, Finetek) and sectioned (20 ␮m) on a cryostat (Hyrax C60, Mi- right to the midline for the recording electrode, and 1 mm posterior to crom). Free-floating sections were blocked with 10% horse serum, 0.2% lambda and 1–2 mm left to the midline for the reference electrode. The bovine serum albumin (BSA), and 0.3% TritonX in PBS for 1 h and then electrodes were fixed with dental cement. Radio transmitters allowed incubated for 12 h (TH) or 48 h (synaptic proteins) in primary antibody simultaneous monitoring of electrocorticogram and motor activity in solution containing 1% horse serum, 0.2% BSA, and 0.3% Triton X-100 undisturbed, freely moving mice that were housed in their home cages. in PBS. For synaptic clusters analysis, antibodies against the presynaptic Telemetry data and corresponding video data were recorded 1 week after marker synaptophysin were coapplied with antibodies against the post- surgery at the earliest and were stored digitally using Ponemah software synaptic scaffolding proteins PSD-95 (excitatory synapse marker) and v5.1 (Data Sciences International). Electrocorticogram recordings lasted gephyrin (inhibitory synapse marker). Primary antibodies used were as over 50 h, sampled at 500 Hz, with continuous video recording. Electro- follows: mouse anti-TH monoclonal antibody (1:5000; IMMUNOSTAR, corticograms were analyzed off-line in MATLAB R2014b (MathWorks). 22941), mouse anti-gephyrin (1:300; Synaptic Systems, 147 011), rabbit To find any ictal activity, we first tried to automatically detect the anti-PSD-95 (1:500; Invitrogen, 51-6900), guinea pig anti-synapto- largest nonpathological electrocorticogram pattern available, namely physin 1 (1:1000; Synaptic Systems, 101004). Sections were subsequently K-complexes. Detection settings for putative K-complexes were defined incubated with fluorophore-conjugated secondary antibodies at room as having a positive and negative peak of at least 2ϫ the amplitude of temperature for 2 h. Secondary antibodies used were as follows: goat baseline activity, at least 15 ms in length each and where the end of a anti-mouse AlexaFluor 555 (1:1000; ThermoFisher, A-21422), goat anti- positive peak necessarily needed to be followed by the start of a negative mouse DyLight 633 (1:200; ThermoFisher, 35513), goat anti-rabbit peak within 15 ms. An additional criterion was to restrict detection dur- AlexaFluor 555 (1:200; ThermoFisher, A-21428), and goat anti-guinea ing slow-wave sleep. For this, the electrocorticogram was Butterworth pig AlexaFluor 488 (1:200; ThermoFisher, A-11073). Sections were then filtered in the delta range (0.5–3 Hz, third order). The instantaneous rinsed and mounted with ProLong Gold Antifade Mountant with DAPI delta amplitude (moving average filter, width 2 s) at any putative (Invitrogen, P36931) and stored in the dark. K-complex needed to be Ͼ95% of the mean instantaneous delta ampli- Brain sections were imaged with a confocal microscope (TCS SP8, tude of the entire recording. Then, any event that exceeded twice the Leica). For quantification of TH immunoreactivity, stacks of 15 images at median peak-to-peak amplitude of these detected putative K-complexes 1 ␮m increment steps were acquired at 20ϫ magnification anda1AU was investigated for possible signs of ictal activity. pinhole. Images were analyzed with FIJI blind to the genotypes. Images For automatic REM-like sleep detection, the electrocorticogram was were background subtracted, and Z-projected to generate a summed- Butterworth filtered in the following frequency ranges: delta (0.5–3 Hz, Gao et al. • Arc/Arg3.1 Involvement in Schizophrenia J. Neurosci., October 9, 2019 • 39(41):8149–8163 • 8153

mM Na3VO4, 1% Triton X-100, 1 mM PMSF, 4 ␮g/ml aprotinin, 1 ␮g/ml leupeptin, and 200 ng/ml pepstatin A in PBS. Protein concentrations were determined using BCA assay (ThermoFisher, Pierce). Equal amounts of protein were electrophoresed on 12% SDS polyacrylamide gels and transferred to PVDF membranes. Western blots were blocked in 5% nonfat milk in PBS with 0.01% Tween and incubated overnight with a polyclonal anti-Arc/ Arg3.1 antibody (1:300,000; Synaptic Systems) or monoclonal anti-GAPDH antibody (1: 500,000; Millipore). After secondary antibody incubation, bands were visualized using Super- Signal chemiluminescence reagent (Thermo- Fisher, Pierce). Immunopositive bands were detected by ImageQuant LAS 4000 (Fujifilm) and analyzed using ImageJ (NIH). Experimental design and statistical analysis. Statistical tests used were as follows: two-tailed two-sample Student’s t test, Mann–Whitney– Wilcoxon test, Kolmogorov–Smirnov test, two-way ANOVA with repeated measures and Bonferroni post hoc test. Type of test is indi- Figure 1. Genetic deletion of Arc/Arg3.1 prenatally or perinatally results in hypoactive PFC networks. A, Illustration of LFP cated in the main text. Statistics were done with recording in the PFC (top) and histological verification of DiI-coated probes (red) with DAPI staining shown in blue (bottom). B, SPSS Statistics 19 (IBM) and OriginPro 2017 Spectrograms of exemplary excerpts of PFC activity for WT, KO, and E-cKO mice. C, D, Reduced theta power in the PFC of KO mice. (OriginLab); p Ͻ 0.05 was considered as signif- E,F,ReducedgammapowerinthePFCofKOmice.G,H,E-cKOmiceexhibitsignificantlyreducedthetapowerinthePFCcompared icant. All graphs were generated with Origin- with their WT littermates. I, J, Significantly reduced gamma power in the PFC of E-cKO mice. Power spectra were calculated from Pro 2017, Igor Pro 6.3 (WaveMetrics), Adobe the LFP during REM-like states. Means are plotted as continuous lines, and shades represent Ϯ SEM. In corresponding boxplots, Illustrator CS5.5, and MATLAB R2014b/R2017b hourglass markers represent the median, 25th and 75th percentiles. Crosses mark outliers. WT: n ϭ 11, KO: n ϭ 10; WT: n ϭ 7, (MathWorks). Values presented in figures are E-cKO: n ϭ 10. Mann–Whitney–Wilcoxon test was applied in D, F, H, and J with *p Ͻ 0.05, **p Ͻ 0.01. mean Ϯ SEM or median with 25th and 75th percentile, as indicated. third order), theta (6–11 Hz, third order), gamma (50–90 Hz, sixth order), and high frequency (150 high-pass, 14th order). The instanta- Results neous amplitudes were further filtered with a moving average filter Deletion of Arc/Arg3.1 before but not during puberty results (width: 2 s), from which a mean was calculated for each frequency band. in reduced prefrontal network activity Putative REM-like epochs needed to meet the following criteria: (1) ex- Schizophrenia is widely considered a neurodevelopmental dis- ceed 65 and 70% of the mean theta and gamma amplitude, respectively; ease, caused by combined genetic deficits and environmental as- Ͻ (2) be 95 and 120% of the mean delta and high-frequency amplitude, saults inflicted during brain development (Rapoport et al., 2012; respectively; (3) show an absence of any detected movement activity; Birnbaum and Weinberger, 2017). Disrupted brain development (4) exceed 7.5 s in length; and (5) be Ͻ120% of the mean high- frequency amplitude 2 min before the putative REM-like epoch. Gaps often results in aberrant brain activity preceding disease onset between detected REM-like epochs Ͻ5 s in length were still deter- and psychosis (Lisman, 2012; Uhlhaas and Singer, 2015; Hunt et mined to be part of a REM epoch. Putative REM-like epochs that were al., 2017). This aberrant activity is particularly evident in the pre- still detected in between bouts of activity were deemed false positives frontal cortex, a region implicated in schizophrenia pathology. and excluded. One power spectrum per mouse was calculated from To examine whether genetic deletion of Arc/Arg3.1 might af- concatenated epochs (nFFT ϭ 4096, nw ϭ 1) and normalized for the fect network activity, we performed LFP recordings from the PFC full range of 0–250 Hz. of urethane-anesthetized WT and KO mice (Fig. 1A). LFP re- ␮ Radioactive in situ hybridization. Sagittal brain slices (14 m) were cordings during REM-like episodes revealed a significant loss of prepared using a cryostat and mounted on slides (Superfrost plus, power in the theta (Mann–Whitney-Wilcoxon test, U ϭ 20, **p ϭ ThermoFisher Scientific). In situ hybridization was performed as de- 0.0076, WT: n ϭ 11, KO: n ϭ 10; Fig. 1B–D) and gamma (U ϭ 20, scribed before (Hermey et al., 2013). A fragment corresponding to nu- ϭ cleotides 2342–2923 of the Arc/Arg3.1 three prime untranslated region *p 0.022; Fig. 1B,E,F) bands in KO mice. (3ЈUTR) was used as a probe and specificity of signals was verified by To investigate whether the reduced network activity reflects a f/f comparing with KO. Probes labeled with [␣- 35S]-UTP were generated developmental effect of Arc/Arg3.1, we first bred the Arc/Arg3.1 according to the manufacturer’s instructions (Promega). Transcribed mice with CaMKII␣-Cre mice (Casanova et al., 2001). The prog- probes were cleaned using G-50 mini columns (GE Healthcare) and eny of this breeding are cKO mice in which Arc/Arg3.1 is irrevers- diluted in hybridization buffer (4ϫ SSC, 50% formamide, 1 M Den- ibly removed between P7 and 14 (Gao et al., 2018). We named hardt’s solution, 10% dextran sulfate, 0.5 mg/ml salmon sperm DNA, these mice early-cKO. Remarkably, like KO mice, adult early- ␮ 0.25 mg/ml yeast t-RNA) to a concentration of 5000 cpm/ l. Mounted cKO mice also displayed significantly reduced theta (U ϭ 68, cryosections of brains were fixed in 4% PFA, acetylated, dehydrated, and **p ϭ 0.00758, WT: n ϭ 7, E-cKO: n ϭ 10; Fig. 1B,G,H) and hybridized at 55°C overnight. Ribonuclease A treatment was performed gamma (U ϭ 64, **p ϭ 0.0027; Fig. 1B,I,J) power in the PFC for 30 min at 37°C. Following a high stringency wash in 0.1ϫ saline sodium citrate buffer at 55°C, slides were exposed to x-ray films (Kodak compared with their WT littermates. Biomax MR; GE Healthcare Bioscience). We further assessed the effects of Arc/Arg3.1 ablation during Immunoblotting. Cortical tissues were dissected and quick-frozen on puberty, on network activity by using a second line of conditional dry ice and stored at Ϫ80°C until used. Dissected tissue was homoge- Arc/Arg3.1 KO mice in which Arc/Arg3.1 was genetically deleted nized in lysis buffer containing 5 mM EDTA, 5 mM EGTA, 50 mM NaF, 1 through CaMKII␣-Cre-mediated recombination starting at P21 8154 • J. Neurosci., October 9, 2019 • 39(41):8149–8163 Gao et al. • Arc/Arg3.1 Involvement in Schizophrenia and completing ϳP36 (Fig. 2A–C; Gao et al., 2018). The progeny of this breeding were named late-cKO mice. In contrast to the constitutive KO and early-cKO, LFP recordings in the PFC of late-cKO mice exhibited normal theta and gamma power, indistinguishable from their WT littermates (theta: U ϭ 64, p ϭ 0.154, gamma: U ϭ 63, p ϭ 0.172, WT: n ϭ 10, L-cKO: n ϭ 10; Fig. 2D–H). Compared with all WT and WT-controls pooled together, KO mice exhibited significantly reduced theta and gamma power (p Ͻ 0.05), whereas late-cKO exhibited un- changed gamma and theta power (p Ͼ 0.05). Collectively, these findings show that expression of Arc/Arg3.1 during early brain development is crucial for establish- ing normal oscillatory network activity in the PFC. Later deletion of Arc/Arg3.1 had no overt effect on network activity in the PFC, similar to our findings in the hippocampus (Gao et al., 2018). Figure2. ConditionaldeletionofArc/Arg3.1lateindevelopmentdoesnotalterPFCnetworkactivity.A,Arc/Arg3.1f/f micewere Altered excitatory and inhibitory bred with Cre recombinase carrying mice to generate L-cKO mice. B, Radioactive in situ hybridization with Arc/Arg3.1 antisense synaptic transmission in the PFC of probe,performedonsagittalsectionsofWTandL-cKObrains.AblationofArc/Arg3.1takesplacebetweenP21andP36incKOmice. KO mice C, Representative Western blots of Arc/Arg3.1 protein from the dissected cerebral cortex of adult WT, KO, and L-cKO mice. D, Alterations in the function, size, or num- SpectrogramsofexemplaryexcerptsofPFCactivityforWTandL-cKOmice.E,F,ComparablethetapowerinWTandL-cKOmice.G, H, Indistinguishable gamma power in WT and L-cKO mice. Power spectra were calculated from the LFP during REM-like states. bers of excitatory and inhibitory synapses Means are plotted as continuous lines and shades represent ϮSEM. In corresponding boxplots, hourglass markers represent the affect network oscillations (Buzsa´ki and median,25thand75thpercentiles.Crossesmarkoutliers.WT:nϭ10,L-cKO:nϭ10.Mann–Whitney–Wilcoxontestwasapplied Wang, 2012) and are linked to schizo- in F and H; n.s., not significant. phrenia and other neuropsychiatric disorders (Uhlhaas and Singer, 2015). To elucidate synaptic mechanisms underlying altered network oscillations in the KO mice we recorded spontaneous and evoked synaptic currents from L5 pyramidal neurons in PFC slices obtained from WT and KO mice (Fig. 3A,B). To measure the strength and temporal overlap of excitation and inhibition, we recorded ePSCs in ACSF and the presence of the type A GABA (GABAA) receptor antagonist gabazine. Example current traces recorded in WT and KO neurons are shown in Figure 3C. In the presence of gabazine, eEPSCs increased in amplitude and duration and displayed additional de- Figure 3. Reduced recurrent excitatory and inhibitory synaptic currents in PFC slices from KO mice. A, Image of a patch-clamp layed peaks, indicative of recurrent excita- recording from L5 neurons in a PFC slice with an extracellular stimulation electrode placed in L1.B, Examples of biocytin-filled and tion. Accordingly, the differential trace reconstructedWTandKOneuronswithsimilardendriticmorphologyandburstfiringpatterns.C,ExamplesofevokedPSCrecorded (eEPSC subtracted from ePSC; Fig. 3C) in ACSF and gabazine and the subtracted eIPSC trace. Stimulus intensity was 50 ␮A. Stimulus artifacts were replaced by dashed reflects predominantly the recurrent in- lines. D, The mean net charge during the ePSC (ACSF) was similar in WT and KO neurons. E, The mean excitatory and inhibitory hibitory current (eIPSC). As exemplified charges were significantly larger in WT neurons compared with KO. F, The E/I charge ratio was not different between WT and KO in Figure 3C, eEPSCs and eIPSCs in WT neurons. Two-way ANOVA with repeated measures was applied in D–F. Symbols show mean Ϯ SEM; n ϭ 6 neurons from 3 mice Ͻ Ͻ neurons were often much larger com- per group; main genotype effect, #p 0.05; Bonferroni post hoc,*p 0.05. pared with KO neurons, although ePSCs recorded in ACSF were comparable. The mean net charge trans- contrast, the mean excitatory and inhibitory charges increased ferred by coactive excitatory and inhibitory synapses (Fig. 3D) abruptly at stimuli Ͼ20 ␮A (WT) and 40 ␮A (KO; Fig. 3E), and increased gradually in both WT and KO neurons and was not were significantly larger in the WT neurons (excitatory charge: ϭ significantly different between them (two-way ANOVA with re- two-way ANOVA with repeated measures, genotype: F(1,10) ϭ ϭ ϭ ϭ Ͻ peated measures, genotype: F(1,10) 0.53, p 0.48; intensity: 5.93, #p 0.035; intensity: F(4,40) 6.79, p 0.001; interaction: ϭ ϭ ϭ ϭ ϭ ϭ ϭ F(4,40) 1.47, p 0.23; interaction: F(4,40) 0.83, p 0.52). In F(4,40) 4.96, p 0.002; Bonferroni post hoc,*p 0.038, 0.022, Gao et al. • Arc/Arg3.1 Involvement in Schizophrenia J. Neurosci., October 9, 2019 • 39(41):8149–8163 • 8155

We, therefore, first examined the number of synapses in the PFC by immunolabeling of the presynaptic marker synaptophysin, the excitatory postsynaptic marker PSD-95 and the inhibitory postsynaptic protein gephyrin (Fig. 4A). La- beling revealed a significant reduction in colocalized (Fig. 4B) and non-colocalized (Fig. 4C) gephyrin clusters in the KO PFC sections (unpaired two-tailed t test, colo- ϭ ϭ ϭ calized: t(70) 2.06, *p 0.043, n 36 ROIs, 3 mice per group; non-colocalized: ϭ ϭ ϭ t(70) 2.01, *p 0.047, n 36 ROIs, 3 mice per group) without a change in the number of PSD-95 clusters (unpaired two- ϭ ϭ tailed t test, colocalized: t(70) 0.18, p 0.86, n ϭ 36 ROIs, 3 mice per group; non- ϭ ϭ ϭ colocalized: t(70) 0.013, p 0.99, n 36 ROIs, 3 mice per group; Fig. 4A–C). Next, we analyzed sEPSCs recorded at Ϫ70 mV (Fig. 4D,E, top traces) and sIPSCs at Ϫ50 mV (Fig. 4D,E, bottom traces). Example traces show a high- frequency of sEPSCs and a lower frequency of sIPSC in both WT and KO neurons (Fig. 4D,E). Cumulative histograms show differences between WT and KO sEPSCs only at the right tail of the distributions, indicating the presence of more events with larger amplitudes (Kolmogorov–Smirnov test, ***p Ͻ 0.001; Fig. 4F), shorter inter-event intervals (IEIs; Kolmogorov–Smirnov test, ***p Ͻ 0.001; Fig. 4G) and faster decay time constants (Kolmogorov–Smirnov test, ***p Ͻ 0.001; Fig. 4H) in the KO neurons. The median values per neuron Figure 4. Altered synaptic properties in the PFC of KO mice. A, Immunolabeling of gephyrin, PSD-95, and synaptophysin in the were not different between WT and KO PFCofWTandKOmiceexhibitreducedgephyrinclustersintheKO.B,C,QuantificationofgephyrinandPSD-95clusterscolocalized for any of the measured properties (un- (B) or non-colocalized (C) with synaptophysin. Gephyrin but not PSD-95 clusters were significantly reduced in the KO mice ϭ Ͻ Ϫ Ϫ paired two-tailed t test, amplitude: t(21) (unpaired two-tailed t test, *p 0.05). D, E, Example traces of sEPSCs and sIPSCs recorded at 70 and 50 mV, respectively, Ϫ ϭ ϭ ϭ 0.99, p 0.33, IEI: t(21) 1.30, p 0.21, from a WT (D) and KO (E) neuron. Boxed regions in the left traces are shown magnified on the right. F–H, Cumulative probability ␶ ϭ ϭ ϭ histograms of the sEPSC and sIPSC amplitudes (F, I), IEI (G, J), and decay ␶ (H, K). Significant changes in the shape of the decay : t(21) 1.60, p 0.13; WT: n 11, distributions were detected for all parameters (Kolmogorov–Smirnov test, ***p Ͻ 0.001). Insets, Averaged median values of the KO: n ϭ 12; Fig. 4F–H, insets). Compari- correspondingbiophysicalproperty.OnlythesIPSCdecay␶showedasignificantreductioninthemedianvalues,withfasterdecay sons of the sIPSCs amplitude and IEIs be- ␶ in the KO (unpaired two-tailed t test, *p Ͻ 0.05). tween WT and KO neurons indicated fewer events with large amplitudes (Kolmogorov– Smirnov test, ***pϽ0.001; Fig. 4I) and lon- 0.019; inhibitory charge: two-way ANOVA with repeated mea- ger IEIs (Kolmogorov–Smirnov test, ***pϽ0.001; Fig. 4J) in the KO ϭ ϭ ϭ sures, genotype: F(1,10) 6.48, #p 0.029; intensity: F(4,40) neurons but equal median values in the WT and KO neurons (un- Ͻ ϭ ϭ ϭ ϭ ϭ 6.69, p 0.001; interaction: F(4,40) 5.34, p 0.002; Bonferroni paired two-tailed t test, amplitude: t(21) 0.86, p 0.39, IEI: t(21) post hoc,*p ϭ 0.033, 0.018, 0.019). Between 25 and 40 ␮A, the Ϫ0.36, p ϭ 0.72; WT: n ϭ 12, KO: n ϭ 11; Fig. 4I,J, insets). In mean excitatory and inhibitory charges in WT were ϳ10- and contrast, the decay time was significantly shorter over the entire dis- 7-fold larger than in KO neuron, respectively. However, the ratio tribution of sIPSCs in the KO neurons (Kolmogorov–Smirnov test, between the excitatory and inhibitory charges (E/I ratio; Fig. 3F) ***p Ͻ 0.001; Fig. 4K). Accordingly, the median values of the sIPSC was similar in WT and KO neurons (two-way ANOVA with re- decay ␶ were significantly reduced compared with the WT (unpaired ϭ ϭ ␶ ϭ ϭ peated measures, genotype: F(1,10) 0.68, p 0.43; intensity: two-tailed t test, decay : t(21) 2.39, *p 0.026; Fig. 4K, inset). ϭ ϭ ϭ ϭ F(4,40) 0.18, p 0.95; interaction: F(4,40) 0.77, p 0.55). Together, these data suggest that the majority of excitatory synapses These data indicate that the excitatory recurrent network gain onto PFC L5 neurons have similar properties in WT and KO mice. In is reduced in PFC slices of KO mice, but the E/I balance is contrast, KO inhibitory synapses have distinctly faster kinetics. preserved. Reduced network gain in the KO slices can be the The reduced PFC activity in the KO mice might result in consequence of decreased synapse numbers, altered synaptic schizophrenia-like behavior. To test this possibility, we subjected properties, or reduced rates of specific recurrent connections both WT and KO mice to a battery of behavioral benchmark tests of within the network. schizophrenia. 8156 • J. Neurosci., October 9, 2019 • 39(41):8149–8163 Gao et al. • Arc/Arg3.1 Involvement in Schizophrenia

Prenatal ablation of Arc/Arg3.1 does not affect social behaviors in adult mice Patients with schizophrenia frequently display abnormal social behaviors, typi- cally expressed by reduced social engagement (Owen et al., 2016). We, therefore, tested Arc/Arg3.1 KO mice in a three- chamber social interaction arena. During the habituation phase, mice were released into the central chamber and allowed to explore the arena for 10 min per day for 2 consecutive days (Fig. 5A). The time spent in each chamber was measured. Mice preferred to explore the two chambers with cylinders and spent comparable time in each chamber (time in chambers left vs ϭ right, paired two-tailed t test, WT: t(8) Ϫ ϭ ϭ ϭϪ 0.75, p 0.47, n 9; KO: t(8) 0.33, p ϭ 0.75, n ϭ 9; Fig. 5B) with no difference between WT and KO mice, suggesting unbiased place exploration. To test the sociability, a dummy mouse made from Lego (Dummy Mouse) and an unfamiliar mouse (Novel Mouse 1) were placed into the cylinders in the left and right chamber, respectively (Fig. 5A). The test mouse was then free to explore all the chambers. Nor- mal sociability is indicated by a clear preference to interact with the unfamiliar mouse (Novel Mouse 1) rather than the object (Dummy Mouse). Analysis of the close interaction time measured as time spent in the immediate vicinity of the chamber confirmed that both WT and KO mice preferably interacted with an unfamiliar living mouse rather than an object (close interaction time Dummy Mouse vs Novel Mouse 1, paired two- ϭϪ ϭ tailed t test, WT: t(8) 6.43, ***p 0.0002; KO: t ϭϪ5.43, ***p ϭ 0.00062; Figure 5. Prenatal ablation of Arc/Arg3.1 does not cause deficits in social engagement, working memory, or sensorimotor (8) gating in adult mice.A, Illustration of habituation, sociability, and social novelty tests in the social interaction arena.B, WT and KO Fig. 5C). No genotype difference was ob- mice spent a comparable amount of time exploring the two chambers with the cylinder, with no place bias during the habituation served in this test (close interaction time phase.C,BothWTandKOmicepreferablyinteractedwiththeNovelMouse1overtheDummyMouseduringthesociabilitytest.D, with Novel Mouse 1 WT vs KO, unpaired Both WT and KO mice preferably interacted with Novel Mouse 2 over Novel Mouse 1 during the social novelty test. All WT: n ϭ 9, ϭ ϭ two-tailed t test, t(16) 1.44, p 0.17), KO: n ϭ 9. E, Illustration of Y-maze test. Visiting of arms in order 1–2-3 is an example of alternation, whereas 1–2-1 of non- indicating normal sociability for KO mice. alternation. F, Similar number of total arm entries in WT and KO mice. G, WT and KO mice made comparable percentage of To assess whether mice could distinguish spontaneous alternations. All WT: n ϭ 10, KO: n ϭ 10. H, Illustration of startle response to 120 dB auditory stimulation. I, a newly introduced mouse from a previ- Illustrationofprepulseinhibitiontest.J,StrongstartleresponseswereobservedinbothWTandKOmiceupon120dBstimulation. ously encountered mouse, mice were sub- K,ComparablePPIinWTandKOmiceatvariousprepulseintensities.L,NodifferenceofmeanpercentageofPPIinWTandKOmice. ϭ ϭ jected to social novelty test in which the All WT: n 10, KO: n 10. Significance was tested with paired t test within groups and two-tailed two-sample t test between Dummy Mouse was replaced with a sec- groupsinB–D.Two-tailedtwo-samplettestwasperformedinF,G,J,andL.Two-wayANOVAwithrepeatedmeasureswasapplied in K. Bars show mean Ϯ SEM; n.s., not significant, *p Ͻ 0.05, **p Ͻ 0.01, ***p Ͻ 0.001. ond stranger mouse (Novel Mouse 2; Fig. 5A). Proper recognition is indicated by Prenatal ablation of Arc/Arg3.1 does not cause a working preference to the second novel mouse. Again, WT and also KO mice spent significantly more time interacting with the Novel memory deficit in adult mice Mouse 2 (close interaction time Novel Mouse 1 vs Novel Mouse Deficits in working memory observed in patients are considered a ϭϪ ϭ ϭ cognitive impairment in schizophrenia (Lett et al., 2014). There- 2, paired two-tailed t test, WT: t(8) 2.61, *p 0.03; KO: t(8) Ϫ4.40, **p ϭ 0.0023; Fig. 5D), suggesting a comparable and in- fore, we assessed mice behavior in the Y maze, which is frequently tact social recognition ability (close interaction time with Novel used to test working memory in rodents (Dember and Fowler, ϭϪ ϭ 1958; Lalonde, 2002; Sanderson and Bannerman, 2012). In this Mouse 2 WT vs KO, unpaired two-tailed t test, t(16) 0.11, p 0.92; Fig. 5D). Notably, because the social recognition test was test, mice are free to move on a three-armed maze and naturally held 10 min after the sociability test, these data also demonstrate alternate between recently visited arms to avoid visiting the same intact short-term memory for the Arc/Arg3.1 KO mice, in agree- arm repeatedly. The percentage of spontaneous alternations ment with our previous report (Plath et al., 2006). serves as a proxy of working memory (Fig. 5E). WT and KO mice Gao et al. • Arc/Arg3.1 Involvement in Schizophrenia J. Neurosci., October 9, 2019 • 39(41):8149–8163 • 8157

Figure 6. Prenatal ablation of Arc/Arg3.1 does not alter the spontaneous locomotor activity and dopaminergic innervation but transiently increases the response to amphetamine. A, Sponta- neous locomotor activity was comparable in WT and KO mice as indicated by total distance moved during 1 h open-field test (WT: n ϭ 12, KO: n ϭ 8). B–F, Locomotor activity following injection of amphetamine (AMP) at concentrations 1.5–2.5 mg/kg body weight. B, Injection of 1.5 mg/kg amphetamine did not increase locomotion in either WT or KO mice. C, Amphetamine at 2 mg/kg elicitedatransientlyincreasedlocomotoractivityinbothWTandKOmice,thatwassignificantlyhigherintheKOmiceduringthefirst5min(WT:nϭ8,KO:nϭ9).D,Comparabletotalpathlength in WT and KO mice during 1 h exploration after injection with 2 mg/kg amphetamine. E, Amphetamine at 2.5 mg/kg elicited a stronger but still transient increase of locomotion in WT and KO mice (WT n ϭ 8, KO n ϭ 9). F, Comparable total path length in WT and KO mice during 1 h exploration after injection with 2.5 mg/kg amphetamine. G, H, Quantification of TH-immunoreactivity shows comparablesignalin(G)prefrontalcortex(WT:nϭ12slicesfrom4mice,KO:nϭ11slicesfrom4mice)and(H)striatumofWTandKOmice(WT:nϭ12slicesfrom4mice,KO:nϭ12slicesfrom 4 mice). Shown are mean Ϯ SEM (A–F), and median, 25th and 75th percentile, min and max value (G, H). Significance was tested with two-way ANOVA with repeated measures in A–C and E. Bonferroni post hoc was performed to test the difference between groups in C and E;*p Ͻ 0.05. Significance was tested with two-tailed two-sample t test between groups in D and F–H; n.s., not significant.

ϭ ϭ Ͻ made similar numbers of arm entries (unpaired two-tailed t test, p 0.45; prepulse intensity: F(4,72) 41.53, p 0.0001; interac- ϭ ϭ ϭ ϭ ϭ WT vs KO: t(18) 0.41, p 0.69, n 10, 10, respectively; Fig. tion: F(4,72) 0.38, p 0.82; Fig. 5K) that were comparable to 5F), indicating a similar exploratory drive. WT and KO mice also their WT littermates (unpaired two-tailed t test, WT vs KO: t(18) exhibited a similar percentage of spontaneous alternations (un- ϭϪ0.78, p ϭ 0.45, n ϭ 10, 10, respectively; Fig. 5L). These data ϭϪ ϭ ϭ paired two-tailed t test, WT vs KO: t(18) 0.91, p 0.37, n indicate that the sensorimotor gating ability is not diminished in 10, 10, respectively; Fig. 5G), indicating intact and indistinguish- the Arc/Arg3.1 KO mice. able performance in this working memory test. Prenatal ablation of Arc/Arg3.1 does not alter spontaneous Prenatal ablation of Arc/Arg3.1 does not affect sensorimotor locomotor activity and only transiently increases the response gating ability in adult mice to amphetamine PPI of the acoustic startle response is frequently used to detect Additional biological traits identified in patients with schizo- sensorimotor gating deficits linked to psychiatric disorders (Braff phrenia are locomotor anomalies (Sano et al., 2012) that are et al., 2001; Swerdlow et al., 2008). Schizophrenia patients repro- traced to hypersensitivity to dopaminergic stimulation (Seeman ducibly display reduced auditory PPI (Mena et al., 2016; Haß et et al., 2005). To test spontaneous locomotor activity, we first al., 2017; Swerdlow et al., 2018). PPI has also been adapted for use subjected Arc/Arg3.1 WT and KO mice to a long-term open field on rodent models as a biomarker of schizophrenia-like pheno- test in which natural exploratory activity was recorded for 1 h. No types (Swerdlow et al., 2008). Given the effectiveness and sensi- difference was observed between WT and KO mice as indicated tivity of this paradigm for schizophrenia phenotype screening, we by a similar path length (two-way ANOVA with repeated mea- ϭ ϭ ϭ assessed the degree of PPI for the Arc/Arg3.1 KO mice. In this test, sures, genotype: F(1,18) 2.37, p 0.14; time: F(10,180) 38.21, Ͻ ϭ ϭ ϭ an intense acoustic pulse alone leads to a strong startle response p 0.0001; interaction: F(10,180) 1.36, p 0.20, WT: n 12, indicated by the peak amplitude of the whole-body acceleration KO: n ϭ 8; Fig. 6A) throughout the testing time, demonstrating measured by a piezoelectric sensor (Fig. 5H). Both WT and KO that KO mice were neither hyperactive nor hypoactive. To test mice generated strong startle responses to the pulse stimulus (120 locomotor activity upon dopaminergic stimulation, we injected dB) with no difference between them, suggesting that KO mice independent cohorts of mice with amphetamine (1.5–2.5 mg/kg had normal auditory perception like WT mice (unpaired two- body weight, i.p.) to evoke dopamine release. Locomotor activity ϭ ϭ ϭ tailed t test, WT vs KO: t(18) 0.22, p 0.83, n 10, 10, respec- was recorded for 10 min before and 1 h after the injection. The tively; Fig. 5J). If a weaker pulse (henceforth the prepulse) is lowest dose of amphetamine (1.5 mg/kg) did not elicit any in- applied before the intense pulse, it leads to a reduced startle re- crease of locomotion in neither WT nor KO mice (Fig. 6B). How- sponse (Fig. 5I). This inhibition effect produced by the prepulse ever, higher doses (2 and 2.5 mg/kg) elicited a transient increase presentation is termed PPI and reflects the sensorimotor gating of locomotion in both genotypes, which was slightly stronger in ability of the mouse (Clapcote et al., 2007). Arc/Arg3.1 KO mice the KO mice during the first 5 or 10 min after injection, respec- ϭ ϭ ϭ consistently showed PPI at different prepulse intensities (two- tively (genotype: F(1,15) 0.32, p 0.58; time: F(11,165) 10.16, ϭ Ͻ ϭ ϭ ϭ way ANOVA with repeated measures, genotype: F(1,18) 0.61, p 0.0001; interaction: F(11,165) 2.84, p 0.002, WT: n 8, 8158 • J. Neurosci., October 9, 2019 • 39(41):8149–8163 Gao et al. • Arc/Arg3.1 Involvement in Schizophrenia

ϭ ϭ KO: n 9; Fig. 6C; genotype: F(1,15) ϭ ϭ Ͻ 2.22, p 0.16; time: F(11,165) 15.24, p ϭ ϭ 0.0001; interaction: F(11,165) 1.31, p 0.23; Fig. 6E; Bonferroni post hoc test, *p Ͻ 0.05, WT: n ϭ 8, KO: n ϭ 9). How- ever, no significant difference was detected in the total path length of WT and KO mice over the 1 h exploration after amphetamine injection (unpaired two- ϭϪ ϭ tailed t test, WT vs KO: t(15) 0.57, p ϭϪ ϭ 0.58; Fig. 6D; t(15) 1.49, p 0.16; Fig. 6F). These data suggest that the spontaneous locomotor activity was not altered in the Arc/Arg3.1 KO mice, and the sensitivity to amphetamine was only transiently increased.

Prenatal ablation of Arc/Arg3.1 does not disrupt dopaminergic innervation in the prefrontal cortex and the striatum To further investigate whether prenatal ablation of Arc/Arg3.1 could affect dopaminergic neurotransmission, we pro- ceeded to directly measure dopaminergic innervation in the PFC (Fig. 6G) and stria- Figure 7. Absence of epileptic brain activity and reduced seizure susceptibility in Arc/Arg3.1 KO mice. A, Two exemplary tum (Fig. 6H). We selected tyrosine spectrograms of a WT (top) and a KO mouse (bottom) show ϳ50 h recording sessions, together with the corresponding activity hydroxylase, the enzyme for catalyzing patterns (below spectrograms). No continuous epileptiform or sharp-wave high-frequency bands were observed in any of the dopamine synthesis as a marker, and per- mice.B,C,Normalcorticalevents(K-complexes)werereadilydetectedinWT(B)andKO(C)mice.Theredhorizontallinesindicate ϫ formed immunofluorescent staining on the detection threshold ( 2 baseline mean), green and red vertical lines indicate the start and stop of the detected events, both WT and KO slices. Dopaminergic fi- respectively.D,SimilarnumbersofputativeK-complexeswerefoundinWTandKOmice.Notethatlargereventsreflectingpossible epileptiform or ictal-like activity were not detected in any of the mice. E, Examples of putative REM epochs are shown. F, Normal- ber intensity was measured from confocal ized power spectra from two WT (black) and three KO mice (red) are shown, all revealing a theta and two separated gamma peaks stack images. Interestingly, the dopamine and no abnormal peaks in the KO. G, A higher dosage of kainic acid is required to evoke generalized seizures in KO mice (WT: n ϭ axon density did not differ between WT 29, KO: n ϭ 21). *p Ͻ 0.05. and KO mice, neither in the prefrontal cortex (Mann–Whitney–Wilcoxon test, and KO mice (Fig. 7B–D). No event, larger than the putative ϭ ϭ ϭ ϭ U 75, p 0.61, WT: n 12 slices from 4 mice, KO: n 11 slices K-complexes, which could reflect inter-ictal activity, was de- from 4 mice; Fig. 6G) nor the striatum (Mann–Whitney–Wilc- tected in any of the mice, confirming that both WT and KO mice ϭ ϭ ϭ oxon test, U 89, p 0.35, WT: n 12 slices from 4 mice, KO: were free of epileptiform or inter-ictal brain activity. We further ϭ n 12 slices from 4 mice; Fig. 6H), suggesting unaltered dopa- analyzed REM-epochs of the recordings to detect possible abnor- minergic projections in the absence of Arc/Arg3.1. mal activity during sleep. Power spectra of two WT and three KO mice all show expected theta and low- and high-gamma bands Absence of epileptic brain activity and reduced seizure (Fig. 7E,F). Moreover, KO mice did not show an additional pro- susceptibility in Arc/Arg3.1 KO mice nounced peak at low frequencies (1–30 Hz), which is typical of Comorbidity of schizophrenia and epilepsy are common in hu- epileptiform and ictal activity (Peters et al., 2005; Shin et al., 2008; mans and animal models (Clarke et al., 2012). To further inves- Jefferys, 2010). We then examined the susceptibility of WT and tigate the possibility that aberrant neural activity and enhanced KO mice to kainic acid-induced seizures. Surprisingly, a signifi- seizure susceptibility might increase the risk of developing cantly higher dosage of kainic acid was required to evoke seizures schizophrenia-like phenotypes in the KO mice, we performed in the KO mice (WT: 27.2 Ϯ 2.1 ␮g/g, n ϭ 29; KO: 35 Ϯ 0.3 ␮g/g, telemetric electrocorticogram recordings combined with video- n ϭ 21; unpaired two-tailed t test, *p ϭ 0.043; Fig. 7G), suggesting and activity-monitoring in freely behaving WT and KO mice for a reduced susceptibility to seizure induction. In summary, our a total duration of 50 h (Fig. 7A–F). No behavioral abnormality, KO mice lacked any sign of epileptiform activity and were, in which could be associated with epileptic activity, was observed in contrast, more resistant to induction of seizures. any of the mice. Moreover, electrocorticograms from individual WT and KO mice (Fig. 7A) show similar patterns of electro- Conditional deletion of Arc/Arg3.1 late in development does graphic activity. Notably, no continuous bands of low-frequency not affect seizure susceptibility, social behavior, or activity were observed in WT or KO mice, indicating the absence amphetamine sensitivity of epileptiform activity. Although PFC activity was intact in late-cKO mice, absence of To investigate the possible occurrence of an inter-ictal activ- Arc/Arg3.1 in the adult brain could reduce brain plasticity, similar ity, such as abnormally large single or multiple spikes in the to knockdown of Arc/Arg3.1 in adult rats (Guzowski et al., 2000; electroencephalogram, we first detected putative K-complexes, Messaoudi et al., 2007) and thereby induce schizophrenia-like which are large biphasic waves normally occurring in the cortex behaviors. To test this possibility, we subjected late-cKO mice to during sleep (Amzica and Steriade, 1997; Marini et al., 2004). several behavioral tests. First, we examined the dosage of kainic Similar numbers of putative K-complexes were detected in WT acid required to evoke generalized seizures in WT and late-cKO Gao et al. • Arc/Arg3.1 Involvement in Schizophrenia J. Neurosci., October 9, 2019 • 39(41):8149–8163 • 8159

Fig. 8F; all WT: n ϭ 8, L-cKO: n ϭ 8). These data further confirm that genetic ablation of Arc/Arg3.1 by itself, before or during puberty, does not cause overt schizophrenia-like behavior in adult mice.

Adolescent social isolation of WT and Arc/Arg3.1 KO mice does not affect social engagement and sensorimotor gating. Studies in animal models show that environmental stressors during adolescence can result in schizophrenia-like behaviors when combined with genetic risk factors (Niwa et al., 2013; Li et al., 2018). We addressed the effect of Arc/Arg3.1 gene- deletion and environment assault by subjecting Arc/Arg3.1 KO mice and WT littermates to social isolation during puberty (Fig. 9A). Isolated adult WT and KO mice spent significantly longer times interacting with Novel Mouse 1 compared Figure 8. Conditional deletion of Arc/Arg3.1 late in development does not affect seizure susceptibility, social behavior, and with Dummy Mouse (paired two-tailed t amphetaminesensitivity.A,ComparabledosageofkainicacidisrequiredtoevokegeneralizedseizuresinWTandL-cKOmice(WT: ϭϪ ϭ n ϭ 12; L-cKO: n ϭ 14). B–C, Sociability and social novelty tests in L-cKO mice. B, Both WT and L-cKO mice preferably interacted test, WT: t(11) 2.99, *p 0.012; KO: ϭϪ ϭ ϭ with Novel Mouse 1 rather than the Dummy Mouse during sociability test. C, Both WT and L-cKO mice preferably interacted with t(6) 2.91, *p 0.027, WT: n 12, KO: NovelMouse2ratherthanNovelMouse1duringsocialnoveltytest.AllWT:nϭ7,L-cKO:nϭ7.D,Spontaneouslocomotoractivity n ϭ 7; Fig. 9B). WT mice, tested for social was comparable in WT and L-cKO mice as indicated by similar path length during 1 h open-field test (WT: n ϭ 8, L-cKO: n ϭ 8). E, recognition, exhibited a significant pref- Indistinguishable locomotor response in WT and L-cKO mice when injected with 2.5 mg/kg body weight amphetamine intraperi- erence to the Novel Mouse 2 (paired two- toneally (WT: n ϭ 8, L-cKO: n ϭ 8). F, Similar total path length was generated in WT and L-cKO mice during 1 h exploration after ϭϪ ϭ tailed t test, WT: t(11) 3.48, **p injection with 2.5 mg/kg amphetamine. Bars show mean Ϯ SEM. Significance was tested with two-tailed two-sample t test 0.0051; Fig. 9C). KO mice exhibited a sim- betweengroupsinA–CandF.PairedttestwasperformedwithingroupsinBandC.Two-wayANOVAwithrepeatedmeasureswas ilar preference, however this did not reach performed in D and E. n.s., not significant, *p Ͻ 0.05. ϭϪ ϭ significance (KO: t(6) 1.46, p 0.19; Fig. 9C). The interaction time with the mice. Unlike KO mice, late-cKO mice developed generalized sei- Novel Mouse 2 was not significantly different between WT and ϭϪ ϭ zures in response to the same dosage of kainic acid as their WT- KO mice (unpaired two-tailed t test, t(17) 0.26, p 0.79). control littermates (unpaired two-tailed t test, WT vs L-cKO: t(24) These data show that isolated WT and KO mice maintain intact ϭ 0.20; p ϭ 0.84; WT: n ϭ 12; L-cKO: n ϭ 14; Fig. 8A), thereby social preference despite prolonged isolation. The same cohort of exhibiting unaltered seizure susceptibility. mice was subjected to the PPI test to probe a second schizo- We next assessed the social behavior of the late-cKO mice to phrenia-like behavior. reveal a possible endophenotype of schizophrenia. Like KO mice, Isolated WT and KO mice generated comparable startle re- late-cKO mice preferred a living mouse over a Dummy Mouse sponses to the pulse stimulus (unpaired two-tailed t test, WT vs ϭ ϭ ϭ ϭ (close interaction time Dummy Mouse vs Novel Mouse 1, paired KO: t(17) 0.10, p 0.92; WT: n 12, KO: n 7; Fig. 9D) and ϭ ϭ ϭ two-tailed t test, WT: t(6) 3.20, *p 0.019; L-cKO: t(6) 2.82, consistently showed indistinguishable PPI rates at different pre- *p ϭ 0.03; close interaction time with Novel Mouse 1 WT vs pulse intensities (two-way ANOVA with repeated measures, ge- ϭϪ ϭ ϭ ϭ ϭ ϭ L-cKO, unpaired two-tailed t test, t(12) 0.91, p 0.38; n 7 notype: F(1,17) 0.55, p 0.47; prepulse intensity: F(4,68) Ͻ ϭ ϭ and 7, respectively; Fig. 8B) and exhibited intact social preference 75.23, p 0.0001; interaction: F(4,68) 1.40, p 0.24; Fig. 9E). to a second novel mouse (close interaction time Novel Mouse 1 vs The mean PPI rate was also similar between WT and KO mice ϭϪ ϭ ϭϪ ϭ Novel Mouse 2, paired two-tailed t test, WT: t(6) 2.94, *p (unpaired two-tailed t test, WT vs KO: t(17) 0.74, p 0.47, ϭϪ ϭ ϭ 0.026; L-cKO: t(6) 2.78, *p 0.032; close interaction time n 12, 7, respectively; Fig. 9F). Additionally, comparison of the with Novel Mouse 2 WT vs L-cKO, unpaired two-tailed t test, t(12) startle responses of socially-isolated mice with the group-housed ϭ 0.16, p ϭ 0.88; Fig. 8C). Additionally, late-cKO mice displayed mice failed to detect any significant difference (all p Ͻ 0.05; data spontaneous locomotor activity similar to their WT littermates not shown). ϭ (two-way ANOVA with repeated measures, genotype: F(1,14) ϭ ϭ Ͻ 1.13, p 0.31; time: F(11,154) 9.71, p 0.0001; interaction: Discussion ϭ ϭ ϭ ϭ F(11,154) 0.72, p 0.72, WT: n 8, L-cKO: n 8; Fig. 8D). Schizophrenia is a strongly heritable neuropsychiatric disease be- Moreover, late-cKO mice did not display hypersensitivity when lieved to arise from polygenetic and environmental risk factors injected with amphetamine (2.5 mg/kg body weight, i.p.), as that converge to disrupt normal brain development and provoke shown by indistinguishable path lengths of WT and late-cKO behavioral, neurological, and cognitive aberrations. Recent stud- mice (two-way ANOVA with repeated measures, genotype: ies identified copy number variations (Kirov et al., 2012; Ferna´n- ϭ ϭ ϭ Ͻ F(1,14) 1.67, p 0.22; time: F(11,154) 10.87, p 0.0001; dez et al., 2017), single nucleotide polymorphisms (Huentelman ϭ ϭ interaction: F(11,154) 0.52, p 0.89; Fig. 8E; total path length et al., 2015), and rare mutations (Fromer et al., 2014; Purcell et ϭϪ ϭ WT vs L-cKO, unpaired two-tailed t test: t(14) 1.29; p 0.22; al., 2014) associated with high schizophrenia risk in genes whose 8160 • J. Neurosci., October 9, 2019 • 39(41):8149–8163 Gao et al. • Arc/Arg3.1 Involvement in Schizophrenia products form complexes with Arc/ Arg3.1. Here we used constitutive and conditional Arc/Arg3.1 KO mice, which we previously generated (Plath et al., 2006; Gao et al., 2018) to investigate the impact of Arc/Arg3.1 deletion on behavioral and neurological domains of schizophrenia. Examination of constitutive Arc/ Arg3.1 KO mice failed to identify any overt deficits in social interaction, working memory, sensorimotor gating, and locomotor activity. Reduced seizure susceptibility and decreased oscillatory activity in the PFC of the anesthetized KO and early-cKO mice are dissimilar to the ex- cessive high-frequency cortical oscillations measured during resting state in schizophrenic patients (Spencer, 2011; Figure 9. Adolescent social isolation does not affect social engagement or sensorimotor gating in adult Arc/Arg3.1 KO mice. A, Hirano et al., 2015). However, reduced Illustration of time flow for social isolation and the following behavioral tests. Mice were socially isolated with individual housing brain oscillations during task perfor- fromP35.B,BothWTandKOmicepreferablyinteractedwiththeNovelMouse1overtheDummyMouseduringthesociabilitytest. mance have also been reported in human C, Both WT and KO mice preferably interacted with Novel Mouse 2 over Novel Mouse 1 during the social novelty test. D, Strong patients and animal models of schizo- startle responses were observed in both WT and KO mice upon 120 dB stimulation. E, Comparable PPI rate in WT and KO mice at phrenia (Lisman, 2012; Uhlhaas and various prepulse intensities. F, No difference of mean percentage of PPI in WT and KO mice. Significance was tested with paired t Singer, 2015). Notably, late-cKO mice, in test within groups, and two-tailed two-sample t test between groups in B and C. Two-tailed two-sample t test was performed in which Arc/Arg3.1 was deleted late in post- D and F. Two-way ANOVA with repeated measures was applied in E. All WT: n ϭ 12, KO: n ϭ 7. Bars show mean Ϯ SEM. n.s., not Ͻ Ͻ natal development, exhibited normal be- significant, *p 0.05, **p 0.01. SI, social interaction. haviors and intact PFC oscillatory activity. We hypothesize that early Arc/Arg3.1 expression in the PFC of Buuse et al., 2011; Schmidt and Weinshenker, 2014), or glutama- late-cKO mice supports the normal formation of synaptic net- tergic transmission (Coyle, 2006). works and maturation of oscillatory activity. Environmental stressors during adolescence are considered The interplay between excitation and inhibition is a key deter- high-risk factors for schizophrenia, especially when combined minant of gain control in recurrent cortical networks (Douglas et with genetic aberrations. Social isolation during adolescence has al., 1995) performing cognitive algorithms such as working been shown to induce depression and schizophrenia-like behaviors in mice harboring genetic mutations linked-to schizophre- memory and perception. The biophysical properties of excitatory nia, but not in WT mice (Niwa et al., 2013; Li et al., 2018). and inhibitory synapses influence oscillatory power and fre- Subjecting Arc/Arg3.1 KO mice to this stressor did not result in quency (Buzsa´ki and Wang, 2012). The faster sIPSCs and reduced deficits in social behavior or PPI. These data indicate that Arc/ gain in the KO PFC may contribute to the impaired oscillations Arg3.1 deletions combined with subthreshold insults do not suf- and learning and memory deficits in the KO mice (Plath et al., fice to manifest schizophrenia-like behaviors. 2006; Gao et al., 2018). The finding that the E/I balance was Our findings are in disagreement with a recent study reporting preserved in the KO PFC can explain why despite changes in overt schizophrenia-related phenotypes in a different Arc/Arg3.1 PFC circuitry and LFP, KO mice do not exhibit schizoph- KO mouse model (Manago` et al., 2016). In this study, Arc/Arg3.1- renia-like symptoms or epilepsy and are, in fact, more resilient deficient mice (EGFP knock-in–Arc/Arg3.1 knock-out), dis- to seizures. In both diseases disrupted E/I balance might be played strongly impaired social interaction and PPI, as well as particularly critical for pathology (Lisman, 2012; Uhlhaas and amphetamine hypersensitivity, and dopaminergic dysregulation. Singer, 2015). In stark contrast, using similar experimental procedures, we Reduced gephyrin clustering and fast decaying sIPSCs could found no apparent schizophrenia-related phenotype in our KO result from a change in subunit composition of GABAA recep- mice. These contradicting observations might be reconciled and tors, which is known to affect receptor kinetics and anchoring explained by the different strategies used for generating the two mechanism at the synapse (Fritschy et al., 2012). Reduced recur- KO mouse models. The mouse model used by Manago` et al. rent excitation to L5 PFC neurons without a concurrent reduc- (2016) was generated by knocking into the Arc/Arg3.1 open read- tion of sIPSC frequency could be a result of a loss of synaptic ing frame (ORF) a d2EGFP sequence terminated by a stop codon currents activated during recurrent but not spontaneous activity, and followed by a floxed neomycin cassette, which has not been for example NMDA receptor currents (Wang et al., 2008). removed in the mutant mice (Wang et al., 2006). The neomycin The normal spontaneous locomotion of the Arc/Arg3.1 KO cassette in these KO mice (EGFP knock-in–Arc/Arg3.1 knock- mice does not recapitulate hyperlocomotion observed in schizo- out) could potentially produce adverse effects (Scacheri et al., phrenic patients (Perry et al., 2010). Moreover, amphetamine- 2001), such as disruption of neighboring gene expression (Pham induced locomotion was far weaker and shorter in the Arc/Arg3.1 et al., 1996), which is not seen in the WT littermates that do not KO compared with other rodent models with relevance to schizo- carry the neomycin cassette. For example, it would be interesting phrenia (van den Buuse, 2010; Young et al., 2016). In the absence to see whether the neomycin cassette could interfere with the of other dopaminergic or behavioral abnormalities, we speculate expression of JRK and LYNX1, two epilepsy-related genes whose that the transient amphetamine response reflects direct or loci are close to the Arc/Arg3.1 gene on mouse chromosome 15 circuit-level modifications in serotonergic, adrenergic (van den (MGI: 88067, NCBI Gene: 11838). This interference might pro- Gao et al. • Arc/Arg3.1 Involvement in Schizophrenia J. 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